Abstract Xenotropic murine leukemia virus-related virus (XMRV) is a newly identified gammaretrovirus linked to prostate cancer and chronic fatigue syndrome (CFS). Furthermore, other murine leukemia virus (MLV)-like sequences more closely related to polytropic MLV have also been reported to be associated with CFS. Since these viruses can be both detected in, and in the case of XMRV directly cultured from, leukocytes and plasma, they are highly likely to be transmissible by blood or blood products. Thus, sensitive, specific and reliable detection of the presence of XMRV, ideally by nucleic acid testing (NAT), is urgently required to further investigate this potentially serious threat to blood safety. Testing will be required initially to fully characterize the pathogenic potential of these viruses, together with the true prevalence in donor populations and the likelihood of transfusion-transmission (TT). These results will then determine whether large scale screening of the blood supply is warranted. Specific Aim 1: Detection of XMRV and other MLV-related viruses. Preliminary results, using validated clinical samples, have demonstrated that NAT detection of XMRV was, at best, variable. However, a 2 to 4 day delay, with samples held at 4oC prior to processing, allowed a more reliable detection of XMRV in the plasma fraction, by multiple laboratories. We hypothesize that regularly processed plasma is not the optimal medium for the detection of XMRV and other MLV-related viruses. Thus in aim 1 we will extend and further investigate these findings. We will perform detailed and extensive comparison of processing methods from multiple XMRV/MLV positive individuals. Plasma that becomes positive after a delay in processing will be treated with nucleases, proteases and detergents before and after ultracentrifugation, to determine whether viral nucleic acid is virion associated or present as free RNA/DNA. Specific Aim 2: Mechanisms to enhance nucleic acid testing. Although the optimization of the delayed processing of plasma will greatly enhance the potential of clinical and basic research studies, it is not likely to be practical for high-throughput screening of blood donors, where screening results are ideally required less than 24 hours after phlebotomy. Thus, we will investigate a multitude of techniques in order to achieve similar results to delayed processing, starting with those most likely to be easily and cheaply achieved in the blood collection setting.